Identification of 7-Deoxy-desulfo-argino-cylindrospermopsin, the Missing Piece in Cylindrospermopsin Biosynthesis

Abstract

Cylindrospermopsin, a major cyanotoxin, is produced by freshwater cyanobacteria. Its biosynthesis starts from arginine and glycine and involves five polyketide synthases and several tailoring enzymes. We report the identification of 7-deoxydesulfo-argino-cylindrospermopsin in several cylindrospermopsin-producing cyanobacteria using mass spectrometry experiments. We have purified this new metabolite and established its structure by 1D and 2D NMR spectroscopy using scalar-based 1 H-1 H, 1 H-13 C, and 1 H-15 N as well as 2D 1 H-1 H ROESY correlation experiments. Using labeled arginines in isotopic incorporation experiments, we have shown that arginine is fully incorporated into 7-deoxy-desulfo-argino-cylindrospermopsin and that the uracil ring of cylindrospermopsin originates from the guanidino moiety of arginine, thus solving a long-standing puzzling question. CyrG and CyrH from the cylindrospermopsin-producing Oscillatoria sp. PCC 6506 were overproduced in Escherichia coli and purified to homogeneity. We showed that CyrG is a zinc-dependent hydrolase, homologous to adenosine deaminases, that transforms 7-deoxydesulfo-argino-cylindrospermopsin into 7-deoxy-desulfo-cylindrospermopsin and ornithine, with the following kinetic parameters: K M = 0.21 ± 0.05 μM and k cat = 0.19 ± 0.02 min −1 . CyrG contained 0.55 mol of zinc per mol of monomer but could be activated by Fe II or Co II . CyrH contained almost no metal and showed no such activity even in the presence of excess metal. Using structurebased alignments and secondary structure predictions, we propose that the fifth and last polyketide synthase CyrF in cylindrospermopsin biosynthesis contains an unprecedented C-terminal domain homologous to N-acetyltransferases. We suggest that this domain catalyzes the condensation of the CyrF product with arginine to give 7-deoxy-desulfo-argino-cylindrospermopsin. This would be an unprecedented termination step for a polyketide synthase.